Ammonium bicarbonate

The sample preparation for mass spectrometry was done as described [4 (link),15 (link)]. Briefly, the samples were denatured in 8 M urea-100 mM ammonium bicarbonate (both Sigma), and the cysteine bonds reduced with 5 mM tris(2-carboxyethyl)phosphine (Sigma) (37 °C, 30 min). The cysteines were alkylated with 5 mM iodoacetamide (Sigma) (22 °C, 60 min), and the samples subsequently digested using sequencing-grade lysyl endopeptidase (37 °C, 2 h) (Wako). The samples were diluted with 100 mM ammonium bicarbonate to a final urea concentration of 1.5 M and followed by digestion using trypsin (Promega) (37 °C, 18 h). Digested samples were acidified with 10% formic acid to a pH of 3.0, and the peptides were subsequently purified with C18 reverse-phase spin columns according to the manufacturer’s instructions (Macrospin columns, Harvard Apparatus). Dried peptides were reconstituted in 2% acetonitrile and 0.2% formic acid prior to MS analyses.

The identity of full-length recombinant lymphostatin was confirmed by in-gel protein digest and peptide analysis. Excised gel-bands were incubated at a porcine trypsin:lymphostatin ratio of ∼1:30, in 50 mm ammonium bicarbonate overnight at 32 °C (Promega). Peptides were identified by matrix-assisted laser desorption ionization (MALDI) mass spectroscopy on a Voyager DE-STR MALDI-TOF mass spectrometer (Applied Biosystems) using an α-cyano-4-hydroxycinnamic acid matrix. The spectral data were processed using Data Explorer software (Applied Biosystems) and the MASCOT NCBInr database searched against the peptide mass map (Matrix Science). To investigate the domain structure of lymphostatin, purified protein was incubated with trypsin at a ratio of 375:1, at 21 °C, to give limited digestion. Aliquots were removed at 1, 2, 3, and 4 h and the reaction stopped by boiling samples adjusted with 2 mm EDTA and 2 mm PMSF in SDS-PAGE loading buffer. Digest products were separated by SDS-PAGE and individual bands were subjected to in-gel tryptic digestion and MALDI-TOF mass spectroscopy as described above. Peptide masses were compared with the sequence of full-length rLifA using GPMAW 9.2 software, mass tolerance 50 ppm (19 (link)). Fragment F1 was purified to homogeneity from other digest products by ion-exchange chromatography (Mono-Q 5/50 GL; GE Healthcare) as described above.

Twenty microgram of purified protein was reduced by adding dithiothreitol (7.5 mM final concentration) in 100 mM ammonium bicarbonate buffer pH ~8, and incubated at 56°C for 45 min. S-alkylation was performed using Iodoacetamide (7.5 mM final concentration). Proteins were precipitated by adding 40 μl per 10 μg protein of ice cold acetone, the pellets washed with 80% acetone, and dried in a SpeedVac vacuum concentrator. The pellets were re-dissolved in 50 mM ammonium bicarbonate and Trypsin (Promega, United States) to 1 g/L and a protein to Trypsin ratio of 60:1. The sample was incubated overnight at 37°C. If required, samples were additionally digested with the endoprotease GluC (Promega, United States). Glycopeptides were then analyzed by capillary reversed-phase chromatography and electro-spray MS using a Agilent Series 6560 LC-INS-QTOF instrument as described previously (Göritzer et al., 2017 (link)).

Protein spots of interest were excised manually and de-stained for 2 × 15 min with 50 mM ammonium bicarbonate (Sigma-Aldrich) solution containing 50% acetonitrile (Sigma-Aldrich). After complete removal of Coomassie dye, gel pieces were dehydrated using 100% acetonitrile. In-gel digestion was carried out by adding 20 µL of freshly prepared trypsin (12.5 ng/µL, Promega Corporation, Madison, WI, USA) to a solution of 50 mM ammonium bicarbonate for 8 h at 4 °C. Digested peptides were removed from the gel by 30 min sonication. The solution was then acidified by the addition of 2 µL of 2% formic acid (Merck-Millipore). The resulting peptide solution was concentrated to 10 µL using a Speed Vac™ vacuum concentrator (1400 rpm for 10–15 min, John Morris Scientific, Chatswood, NSW, Australia) and stored at -80 °C for future use or immediately subjected to LC/MS/MS [52 (link)].

To ensure global and quantitative (by SWATH-MS) protein identification, an equal amount of proteins from the treated patients and controls were loaded on a 10% SDS-PAGE gel. The run was stopped as soon as the front had penetrated 3 mm into the resolving gel [59 (link),60 (link)]. The protein band was detected by Sypro-Ruby fluorescent staining (Lonza, Basel, Switzerland), excised, and processed for in-gel and manual tryptic digestion, as described [61 (link)]. Gel pieces were reduced with 10 mM dithiothreitol (Sigma-Aldrich, St. Louis, MO, USA) in 50 mM ammonium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) and alkylated with 55 mM iodoacetamide (Sigma-Aldrich, St. Louis, MO, USA) in 50 mM ammonium bicarbonate. Then, the gel pieces were rinsed with 50 mM ammonium bicarbonate in 50% methanol (HPLC grade, Scharlau, Barcelona, Spain), dehydrated via the addition of acetonitrile (HPLC grade, Scharlau, Barcelona, Spain), and dried in a SpeedVac. Modified porcine trypsin (Promega, Madison, WI, USA) was added to the dry gel pieces at a final concentration of 20 ng/μL in 20 mM ammonium bicarbonate, incubating them at 37 °C for 16 h. The peptides were extracted thrice by 20 min incubation in 40 μL of 60% acetonitrile in 0.5% formic acid. The resulting peptide extracts were pooled, concentrated in a SpeedVac (TermoFisher Scientific: Haysham, Lancashire), and stored at −20 °C.

Bands from SDS–PAGE gel were cut out with a scalpel and destained in 30% (v/v) ACN (Merck: 1.00029.1000), 25 mM ammonium bicarbonate (Sigma: 09830) and incubated in an ultrasonic bath for 30 min. This step was repeated until all gel pieces appeared clear. Afterwards, the gel pieces were dehydrated using 100% (v/v) ACN for 15 min at room temperature. The acetonitrile was removed, and proteins were reduced and alkylated using 10 mM TCEP (Sigma: C4706) and 25 mM 2‐chloroacetamide (CAA, Sigma: C0267) in 50 mM ammonium bicarbonate for 45 min at room temperature in the dark. Gel pieces were dehydrated afterwards as described above, and supernatant was removed. Trypsin/LysC (Promega: V5111) was resuspended in 100 mM ammonium bicarbonate to a concentration of 0.05 μg/μl, and 20 μl (= 1 μg) was added to each vial containing the dehydrated gel pieces. Acetonitrile was added to a final concentration of 10% (v/v), and protease digest was carried out overnight at 37°C and 300 rpm using a Thermomix (Eppendorf). Peptides were extracted in three steps: 1% (v/v) FA (Merck: 5.33002.0050); 60% (v/v) ACN, 1% (v/v) FA; and as last step 100% (v/v) ACN. Extraction was carried out for 15 min using an ultrasonic bath. After each step, the respective supernatant was removed, combined into a tube and freeze‐dried (ScanVac CoolSafe) overnight.